Researchers have acknowledged for decades that the small-molecule restoration of wild type p53 function in tumors is one of the ultimate, yet elusive goals in cancer drug development. TP53 is the most commonly mutated gene in cancer and loss of its function is one of the central drivers of tumorigenesis. The majority of mutations are missense and generate a defective protein found at high levels in cancer cells. The p53-R175H is the most common missense mutant and is misfolded because the substitution impairs the protein's ability to bind zinc. We discovered a zinc chelating small-molecule (ZMC1) that can restore wild type structure/function to the p53-R175H by restoring zinc-binding and facilitating proper folding (so-called zinc metallochaperone (ZMC) mechanism). ZMC1 reactivates mutant p53 and selectively kills cancer cells by a p53 mediated apoptotic program both in vitro and in vivo. The pharmacologic restoration of a folding defect in a metalloprotein by metal ion delivery is unprecedented in drug development. We found that other zinc chelators with a similar affinity (Kd) for zinc can function as ZMCs and ZMCs can reactivate other mutants with impaired zinc-binding. Therefore, we hypothesize that ZMCs can be developed as effective mutant p53 targeted anti-cancer drugs. This will be investigated through the following specific aims: 1) Determine the impact of zinc homeostatic mechanisms on ZMC1 pharmacodynamics. Cells respond to the ZMC1 surge in zinc levels by normalizing zinc through homeostatic mechanisms. We hypothesize that these mechanisms regulate ZMC1 activity. We will explore this through mechanistic studies that measure the effect on ZMC1 activity of manipulating zinc regulatory genes in tumors cells. 2) Define the p53 mis-sense mutational spectrum that is amenable to ZMC reactivation. ZMC1 is proposed to reactivate the mutant class with impaired zinc binding; however the full spectrum of this class is unknown. We hypothesize that the mutants closest to the zinc-binding pocket are most likely to have impaired zinc binding. We will study the 15 most frequent mutants within 10 of the zinc-binding pocket. This will define the potential patient poo for ZMCs. 3) Design a ZMC optimized for potency, toxicity, and efficacy in vivo. We have designed novel chemotypes that have ZMC activity and are more potent than ZMC1 in vitro. We will validate these results in vivo. Our team is composed of three laboratories that are leading this area of research. Their expertise is the following: 1) p53 biology (Darren Carpizo), Zinc/p53 folding biophysics (Stewart Loh), and zinc chelator drug design (David Augeri). Relevance to public health: The research performed in this proposal will provide the foundation for the development of a new class of anti-cancer drugs that target the most commonly mutated gene in human cancer. These drugs will have broad activity against all types of cancers.